This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell migration is a fundamental process in biology, crucial for cancer metastasis, embryonic development, wound healing and many other biological processes. The overall aim is to study the migration and invasion of tumor cells using novel microscopy techniques. This is an important biological problem with potential consequences in many areas of medical and clinical interest. Specifically, this research will seek to establish how to use "pair correlation approaches" to study cellular adhesions in 3D tissue assemblies. We studied the migration of individual tumor cells, derived from the human glioma cell line ACBT that were implanted into a 3D gel matrix of collagen type I. Depending on the density of the matrix, cells can migrate in this 3D tissue model. The properties of the matrix are crucial for the migration of the cells. The cells and the collagen fiber network where imaged using a confocal microscope in the reflection mode so that the sample could be observed for a long period of time without bleaching. We developed a method based on image correlation spectroscopy and on local measurement of thermal fluctuations to determine the average size and the local stiffness of the collagen fibers. Intensity fluctuations were generated by the flickering of the reflections from the fibers. The reflected intensity from a single point in the fiber fluctuates due to fiber position fluctuations. We found a correlation between the fiber diameter and the fluctuation spectrum. Using continuous acquisition for several hours we determined the displacement of the fibers close to the cell as the cell forces its way in the 3D network during the migration process. We also observed changes in the fluctuation spectrum at specific locations probably due to the formation of cellular adhesions.